MURI Pattern Detection Circuits Andrew Yang, Jonathan Babb, Ron Weiss 9/23/2011

Null versus 1 Systems (Circuits #1-6) “1” Circuit operation: Initial state of toggle set for CI production with IPTG. Later, UV induces state change for a region by cleaving CI. If the region is large enough to achieve an engineering quorum, the quorum is amplified to the entire region and reported by a blue fluorescent protein. Goal: Digital behavior. Circle sizes will be smaller or larger than a forbidden band. UV Toggle 3OC12HSL Quorum Sensor 3OC12HSL Propagator State “1” = 30C12HSL NOTE: Not depicted is a stretch of DNA following the promoters and preceding the genes called the ‘RBS’. The RBS is tunable, thus controlling the strength of gene expression. Please account for this possibility.

Circuit #1 Las System UV Quorum Sensor Propagator Toggle Switch pLasORI LasI LasR mCherry LacI pLac pCI UV 30C12HSL IPTG pLas GFP Quorum Sensor Propagator Toggle Switch Quorum sensor activated only in UV exposed region.

Circuit #2: External atc->LasR 30C12HSL 30C12HSL TetR pLacIq IPTG UV Atc applied regions produce lasr. QS lasr activated in exposed regions. LacI pCI pLac CI pLasORI LasI LasR mCherry pLas LasI GFP ptet LasR Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.

Circuit #3: atc->LasI Activator 30C12HSL atc 30C12HSL IPTG UV pLas Activator pLacIq TetR Additional activator (cascade) added from circuit 2 for rightward shift of transfer curve. LacI pCI pLac CI pLasORI LasI LasR mCherry pActivator LasI GFP ptet LasR Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.

Circuit #4: Const. Lactonase atc->LasR TetR pLacIq LasR ptet 30C12HSL IPTG UV 30C12HSL Lactonase establishes AHL degradation baseline for rightward shift in transfer curve. LacI pCI pLac CI pLasORI LasI LasR mCherry pLas LasI GFP pConstitutive AiiA Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.

Circuit #4A: Const. Lactonase No atc LasR pLacIq AiiA 30C12HSL IPTG UV 30C12HSL RBS tuning lactonase LacI pCI pLac CI pLasORI LasI LasR mCherry pLas LasI GFP Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.

Circuit #5: atc->Lactonase Positive Feedback LasI TetR pLacIq ptet AiiA 30C12HSL 30C12HSL IPTG UV LacI pCI pLac CI pLasORI LasI LasR mCherry pLas LasR LasI GFP Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.

Circuit #6: atc->Inducible Lactonase Positive Feedback LasI TetR pLacIq pLas/tetO AiiA 30C12HSL IPTG UV 30C12HSL LacI pCI pLac CI pLasORI LasI LasR mCherry pLas LasR LasI GFP Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.

Null versus 1 versus 2 Systems (Circuit #7) “1” “2” Key: LasR has low sensitivity and only detects large circles (2) while RhiR system has high sensitivity for small circles (1). TetR represses small circles if when large are also present. UV Toggle 3OC12HSL Quorum Sensor 3OC12HSL Propagator C4HSL Quorum Sensor C4HSL Propagator State “1” = C4 ^ C12 State “2” = C12 Null = NOR(C4,C12) Operations: now we have two separate quorum sensing systems tuned to different sensitivities. To detect large circles, LasR will amplify with a low sensitivity. In the case where there is no large circle, a more sensitive RhiR system will detect and amplify a smaller circle. Forbidden bands in between each range are not unused by the digital input. NOTE: As before, not depicted is a stretch of DNA following the promoters and preceding the genes called the ‘RBS’. The RBS is tunable, thus controlling the strength of gene expression. Please account for this possibility.

Circuit #7: Null versus 1 versus 2 system C4HSL CI pLasORI LasI LasR mCherry LacI pLac pCI UV 30C12HSL IPTG pLas BFP Quorum Sensors Propagator Toggle Switch C4HSL pRhiORI RhiI RhiR mCherry pRhi RhiI RhiR GFP

Appendix Following are circuit #1 with the Rhil and Lux systems. The same changes in promoters, inducers, and repressors can be applied to circuits #2-6.

Circuit #1 Rhil System UV Toggle Switch Quorum Sensor Propagator C4HSL C4HSL IPTG UV LacI pCI pLac CI pRhiORI RhiI RhiR mCherry pRhi RhiI RhiR GFP Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.

Circuit #1 Lux System UV Toggle Switch Quorum Sensor Propagator C6HSL C6HSL IPTG UV LacI pCI pLac CI pLuxORI LuxI LuxR mCherry pLux LuxI LuxR GFP Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.